Transfer RNAs are essential in all domains of life for translating the information contained in the genetic code into proteins. Recently, fragments of tRNAs, known as tRFs, have been discovered and recognized as non- random products obtained from specific processing of tRNAs and their precursors. tRF-1s, the 3' trailer fragment, are clearly produced by ELAC, and tRNA halves appear to be generated by angiogenin, but it is unclear how the tRFs from the 5' end (tRF-5s) and those from the 3' end (tRF-3s) are generated. Although biological functions for a few tRFs have been reported, the role of tRFs in the cell is unknown. miRNAs are in similar abundance and are of a similar size to tRFs but are generated by drosha and dicer RNAses and function by associating with Argonaute proteins to target mRNAs for degradation. miRNA-mediated RNA silencing plays an important role in many cellular processes and diseases. In this proposal, we will use the existing RNA-Seq data generated after PAR-CLIP and CLASH of Argonaute proteins to demonstrate that tRFs participate in RNA silencing. This analysis will lead to high value targets of tRFs, which will be experimentally validated. We will then use a drosha- and dicer-negative cell line to generate our own RNA-Seq data from PAR-CLIP and CLASH of Argonaute proteins. With miRNAs absent from the cell, there will be an enrichment in tRF reads associated with Argonaute proteins and we will be able to exhaustively identify the cellular RNAs that associate with and are regulated by tRF-Argonaute complexes. These results will be the first to show a clear function for tRFs and could result in the discovery of a role for tRFs in different diseases.